Decontamination of animal feed containing prion (eg. BSE agent)

ABSTRACT

A method of producing an animal feed that is free from transmittable degenerative encephalopathies is provided. Central to the method is alkali treatment of animal material at a pH of at least 8.5 under temperature conditions below 100 C at atmospheric pressure. This method provides a decontaminated animal feed produced under relatively low temperature and pressure conditions that are achievable in standard animal carcass rendering facilities.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.10/416,602, filed Sep. 15, 2003 (now U.S. Pat. No. 7,618,673), whichclaims priority from PCT International Appln. No. PCT/AU2001/001474,filed Nov. 14, 2001 and corresponds to Australian Application No. PR1527, filed Nov. 15, 2000 which claims benefit of U.S. ProvisionalPatent Application Ser. No. 60/286,341, filed Apr. 26, 2001. The presentapplication claims priority to the aforementioned patent applications,which are incorporated in their entirety herein by reference for allpurposes.

FIELD OF THE INVENTION

This invention relates to a method of producing an animal feed. Moreparticularly, this invention relates to a method of producing an animalfeed from animal by-products where treatment with alkali and heateliminates or reduces transmittable degenerative encephalopathies suchas Bovine Spongiform Encephalopathy, Creutzfeldt-Jacob Disease andscrapie.

BACKGROUND OF THE INVENTION

Transmittable Degenerative Encephalopathies (TDE) include bovinespongiform encephalopathy (BSE or “mad cow disease”), scrapie in sheep,Creutzfeldt-Jacob Disease (CJD), Gerstman-Straussler Schemker (GSS) andKuru in humans. These diseases have achieved considerable notoriety inrecent years at least partly due to the perception that authoritiesfailed to monitor incorporation of BSE-contaminated beef into human andanimal food supplies, which led to outbreaks of CJD, mainly in theUnited Kingdom (see Editorial in Nature, 1997, 389 423).

It is now well established that the agent responsible for TDEtransmission is a protein, generally referred to as a “prion” protein,which underlies both BSE and CJD (Hill et al., Nature 1997, 389 448).

Generally, it has been concluded that the destruction of TDE in meatrequires treatment at 132° C. for 20 minutes under 3 bars pressure.Alternatively, in the presence of alkali the temperature and pressuremay be reduced to 121° C. and 2 bars respectively.

Alkali is known for its hydrolytic effect upon biomolecules such asproteins, and efforts to sterilize animal tissue contaminated with TDEhave utilized alkali treatment, heat and pressure as means fordestroying prion pathogenicity.

For example, Taguchi et al., 1991, Arch. Virol. 119 297 used a 1 hourtreatment with 1 N NaOH followed by autoclaving at 121° C. for 30minutes to inactivate CJD-infected brain homogenates. Ernst & Race,1993, J. Virol. Methods 41 193 employed autoclaving together with NaOHand LpH treatment inactivate scrapie-infected brain homogenates.

A more extensive series of tests was performed by Taylor et al., 1994,Arch. Virol. 139 131 to decontaminate BSE-infected bovine brain orscrapie-infected rodent brain samples. Treatments included 1 M or 2 MNaOH for up to 1 hour, autoclaving at temperatures between 134° C. and138° C. for up to 1 hour or treatment with sodium hypochlorite or sodiumdichloroisocyanurate for up to 2 hours. These authors concluded thatnone of the procedures tested produced complete TDE inactivation.

The problem for manufacture of animal feed from potentially TDE-infectedanimal tissue is that high temperature and pressure conditions are notreadily achievable using standard rendering or animal waste recyclingequipment. Also, high temperature treatments during manufacture ofanimal feed tend to produce an inferior feed having unwanted by-productssuch as carcinogens, di-amino acids and volatile odors.

OBJECT OF THE INVENTION

It is therefore an object of the invention to provide a method ofmanufacturing animal feed where the potential for TDE contamination isat least reduced, if not eliminated.

SUMMARY OF THE INVENTION

In one aspect, the present invention resides in a method ofmanufacturing an animal feed including the steps of:

-   -   (i) adding alkali to animal material to maintain a pH of at        least 8.5;    -   (ii) heating the material at step (i) to a temperature in the        range 55° C. to 99° C.; and    -   (iii) dehydrating the material produced at step (ii).

In another aspect, the present invention resides in a method ofmanufacturing a TDE-decontaminated animal feed including the steps of:

-   -   (i) adding alkali to TDE-contaminated animal material to        maintain a pH of at least 8.5;    -   (ii) heating the material at step (i) to a temperature in the        range 55° C. to 99° C.; and    -   (iii) dehydrating the material produced at step (ii).

Suitably, the method of the invention is performed at about atmosphericpressure.

Preferably, sufficient alkali is added to maintain a pH of at least 9.5.

More preferably, sufficient alkali is added to maintain a pH in therange 10.5 to 13.0.

Even more preferably, sufficient alkali is added to maintain a pH in therange 11.0-11.5.

Preferably, the alkali is calcium hydroxide such as in the form ofhydrated lime.

Preferably, the material at step (ii) is heated to a temperature in therange 60° C. to 90° C.

In one particular embodiment, the temperature is about 60° C.

In another particular embodiment, the temperature is 80° C.-85° C.

The duration of steps (i) and (ii), termed the “hydrolytic phase”, ispreferably 1 to 4 hr or more preferably 1 to 2 hr.

After this hydrolytic phase, the treated animal material may be storedprior to dehydration or immediately dehydrated.

With regard to dehydration, it is preferred that the moisture content ofthe animal feed is no greater than 10-15 wt %. Typically, with time thelevel of moisture may drop to about 7-8 wt % which is optimal for theanimal feed of the invention.

In yet another aspect, the invention provides an animal feed producedaccording to the process of the first-mentioned aspect.

In still yet another aspect, the invention provides a TDE-decontaminatedanimal feed produced according to the process of the second-mentionedaspect.

The invention also provides a TDE-decontaminated animal materialproduced according to steps (i) and (ii) of the second-mentioned aspectof the invention.

The invention also extends to use of the animal feed of theaforementioned aspects as a fertilizer.

Throughout this specification, unless otherwise indicated, “comprise”,“comprises” and “comprising” are used inclusively rather thanexclusively, so that a stated integer or group of integers may includeone or more other non-stated integers or groups of integers.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

Table 1: Final pH of variously treated meat samples.

Table 2: Summary of prion protein clearance rates of TDE-spiked meatsamples after alkali and heat treatment.

FIG. 1: Example of apparatus for producing an animal feed.

FIG. 2: Titration of 263K stock SP0172200. Lane 1: Proteinase K digested263K Hs stock, 10^(−2.1). Lane 2: Undigested 263K Hs stock, 10^(−2.1).Lane 3: Low molecular weight rainbow markers. Lanes 4-10: Serial 5-folddilutions of proteinase K digested 263K stock from 10^(−1.4) to10^(−5.6) dilution, respectively.

FIG. 3: Analysis of process samples following proteinase K digestion.Lane 1: Proteinase K digested 263K Hs stock, 10^(−2.1), Lane 2: LowMolecular ratio rainbow markers. Lane 3: Proteinase K digested 287.1A.Lane 4: Proteinase K digested 287.1-1. Lane 5: Boiling Mix 1×. Lane 6:Boiling Mix 1×. Lane 7: Proteinase K digested 287.1-2. Lane 8:Proteinase K digested 287.1-3. Lane 9: Proteinase K digested 387.1-4.Lane 10: Boiling Mix 1×.

FIG. 4: Analysis of undigested process samples. Lane 1: Undigested 263KHs stock, 10^(−2.1), Lane 2: Proteinase K digested 263K Hs 30 stock,10^(−2.1). Lane 3: Undigested 287.1A. Lane 4: Undigested 287.1-1. Lane5: Low Molecular ratio rainbow markers. Lane 6: Boiling Mix 1×. Lane 7:Undigested 287.1-2. Lane 8: Undigested 287.1-3. Lane 9: Undigested287.1-4. Lane 10: Boiling Mix 1×.

FIG. 5: Titration of 263K stock SP0172200. Lane 1: Proteinase K digested263K Hs stock, 10^(−2.1). Lane 2: Undigested 263K Hs stock, 10^(−2.1).Lane 3: Low Molecular ratio rainbow markers. Lanes 4-10: Serial 5-folddilutions of proteinase K digested 263K stock, from 10^(−1.4) to10^(−5.6) dilution, respectively.

FIG. 6: Western blot analysis of proteinase K-digested samples. Lane 1:positive control at 10^(−2.8) dilution. Lane 2: molecular weightmarkers. Lane 3: sample 309.1A. Lane 4: blank. Lanes 5-10: samples309.1-1.3, 309.1-1.6, 309.1-2.3, 309.1-3.3, 309.1-4.3 and 309.1-4.3,respectively.

FIG. 7: Western blot analysis of proteinase K-undigested samples. Lane1: positive control at 10^(−2.8) dilution. Lane 2: molecular weightmarkers. Lane 3: sample 309.1A. Lane 4: blank. Lanes 5-10: samples309.1-1.3, 309.1-1.6, 309.1-2.3, 309.1-3.3, 309.1-4.3 and 309.1-4.3,respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention arises from the unexpected discovery by thepresent inventor that alkali-treated animal material subjected to mildheat treatment at atmospheric pressure will effectively destroy TDE.Minimizing heat treatment is also relevant to the fact that the animalfeed must be highly digestible, particularly with respect to proteincontent, as well as being TDE free. Furthermore, excessive heat willgenerate unwanted products such as cross-linked amino acids,racemization of L-amino acids to their D-isomers and formation ofmutagens such as 2-amino 3,8-diethylimidazole[4,5f]quinoline. Also, itis important that minimal volatilization occurs during manufacture, sothat noxious odor production is minimized. To these ends, the presentinvention minimizes heat treatment during manufacture at atmosphericpressure while utilizing alkali and dehydration steps to effectivelysterilize the animal feed with respect to TDE. The manufacturingconditions provided by the present invention are readily within thecapabilities of many standard commercial animal feed manufacturingfacilities, or can be utilized with only minor modification of suchfacilities.

The animal materials which may be used include, for example, animalwastes and offals from slaughter works; domestic animals of littlecommercial value such as cast for age or drought-affected sheep orcattle, or flock reduction sheep; waste or discarded wildlife carcassesor parts thereof; poultry offal or cast for age poultry; fish orcrayfish offals or non-usable species from catches.

The animal material is preferably mixed in bulk with a dry dehydratingmaterial which is capable of absorbing moisture (either chemically orphysically) from the animal material, reducing the percentage of watercontent in the material to a dry stable product.

The dehydrating materials include at least one or more of thecombinations of:

bentonites, zeolites, kaolins or other clays in a ratio not exceeding35% (w/w);

calcium oxide, magnesium oxide or aluminium oxide in a ratio notexceeding 35% (w/w);

diatonmite or other diamataceous earths in a ratio not exceeding 35%(w/w);

gypsum, dolomite, limestone, sodium bicarbonate or salt in a ratio notexceeding 35% (w/w);

calcium phosphates and/or phosphoric acid in a ratio not exceeding 35%(w/w);

ferrous sulphate and/or ferric sulphate in a ratio not exceeding 35%(w/w);

grains, starches and gelatinous materials and byproducts of grains (forexample, pollard, bran, hulls and the like) including extruded forms ina ratio not exceeding 80% (w/w);

protein, grains and oilseed grains and their byproducts includingprocessed and extruded forms of protein meals in a ratio not exceeding80% (w/w);

vegetable products and byproducts such as copra meal and palm kernelmeal, gin trash and chopped hay and straw in a ratio not exceeding 75%(w/w); and

animal byproducts such as meat meals, bone meals and blood meals andgelatinous materials in a ratio not exceeding 75% (w/w).

The preferred dehydrating materials which can be used are varied andwill depend on several factors including:

-   -   1. The proximity and cost of the dehydrating material to the        dehydrating facility.    -   2. The rate at which the dehydrating needs to be carried out.    -   3. The intended use of the resultant dried product.

Dehydration is preferably performed in a rotary drier.

The alkali used at step (i) may include oxides, hydroxides and salts ofthe metallic elements. Examples include calcium oxide, calciumhydroxide, sodium hydroxide, sodium carbonate, sodium sulphite,potassium hydroxide, potassium carbonate, magnesium hydroxide, magnesiumcarbonate, magnesium sulphate or any two or more in combination.

Preferably, the alkali is in the form of calcium hydroxide (hydratedlime).

The concentration of alkali used at step (i) will depend on the desiredpH, the particular alkali used and the buffering capacity of the animaland other materials used during manufacture.

For example, a typical ratio is 25 kg hydrated lime to 1200 kg wetanimal material.

Dependent on the material with which the product is to be mixed, the pHlevel may be adjusted by the addition of acid.

During manufacture, optional additional materials may be added whichwill enhance the nutritional or economic value of the finished product.Such additional materials (although not limited to) may include, forexample, rumen modifiers such as monensen or avoparcin, enzymes orbacterial cultures; additional vitamins or minerals; non proteinnitrogen sources such as urea; antioxidants, stabilizers, antibiotics,mould inhibitors, preservatives (including salt) and the like; proteinand lipid modifiers to alter their rumen digestibility; palatabilityenhancers such as molasses and byproducts of molasses fermentation.

Preferably, following drying the animal feed is left to stand for 24hours before being fed directly to livestock (both ruminants andmonogastrics) in a granular or block form or as a milled fine powder.

The animal feed may be mixed with feed supplements, trace elements,protein meals, cereal protein and oilseed grains, molasses or byproductsof molasses fermentation, hay or the like in any combination forlivestock (both ruminant and monogastric) feeding; it may be used as apet food either as produce or mixed with other materials; and/or it maybe used directly or as an ingredient in food for human consumption.Generally, the dried material will be mixed at a maximum of 10-15% (w/w)of the final product.

Some examples of the manufactured animal feed are:

-   -   1. Defatted abattoir solids fractions (typically <10% fat on a        dry matter basis) plus stickwater are mixed with calcium        hydroxide to give the required pH of 11 (approximately 1:30 w/w        on a dry matter basis) and allowed to stand in a surge bin for 1        hour. The product is then dried at 80° C. in a rotary dryer over        1 hour to give a resultant product of <10% free moisture which        is then milled to a fine meal.    -   2. Defatted abattoir solids fractions (typically <10% fat on a        dry matter basis) are mixed with calcium hydroxide to give the        required pH of 10 (approximately 1:50 on a dry matter basis) and        allowed to stand in a surge bin for 30 minutes and then dried in        a rotary drier with some solar heat input over 3 hours at 60° C.        to give a resultant product of <10% moisture. This product is        then milled to a fine meal.    -   3. Raw waste fish material is mixed with alkali consisting of        80% calcium hydroxide and 20% sodium hydroxide to a pH of 10.5        (approximately 1:40 on a dry matter basis) and dried in a rotary        dryer at 70° C. over 4 hours to give a resultant product of <11%        moisture which is then milled to a fine meal.

Although, there is yet to be detected any infective TDE in domesticanimals other than ruminants (for example fish and poultry), there arebans on the feeding of protein meals of broad animal origin in somecountries. For example, in Australia and the U.S. there is a ban on thefeeding of any protein meal of animal origin to ruminant animals. Inmany European countries, there is a total ban on the feeding of anyprotein meal of animal origin to all animal species. These bans existfor the fear of infective TDE eventually being detected in animals otherthan ruminants.

It will also be appreciated that the animal feed of the invention may beused as a TDE-decontaminated fertilizer.

To enable the invention to be fully understood, preferred embodimentswill now be described with reference to the accompanying drawing(FIG. 1) which is a schematic diagram of the apparatus employed in themethod of the present invention.

With regard to the method of the invention, the dehydrating material ismixed with the raw animal material and then heated and dried. It isusual to mix all materials at the start but not essential as anyingredient can be introduced at any time during the process. Once mixed,the material is then dried. Any heated drying system may be used but anormal heated air flow system is usually most practical.

Referring to FIG. 1, animal wastes 10 (e.g. cattle carcasses) are passedthrough a mincer or hogger 11 before alkali 12 (preferably calciumhydroxide) is added and mixed in mixer 13.

After mincing and before addition of alkali 12, animal waste 10 may beheated to liquefy the tallow component. The heated animal waste is thendecanted or pressed to create a solid and liquid fraction. The liquidfraction is acidified and separated into a tallow fraction, otherliquids (mostly water) and residual solids (referred to as“stickwater”). This stickwater may be processed separately or combinedwith the solid fraction for alkali and heat treatment.

In this example, addition of alkali 12 (preferably calcium hydroxide)raises the pH of the mixture to about pH 11.0-11.5. During this“hydrolytic phase”, the pH should be maintained at least at 8.5 orpreferably pH 9.5 for 1-4 hrs. The pH may fall as protein hydrolysisoccurs, so it is preferable to start at a pH of at least 11.0 so thatthe pH never falls below pH 8.5 or preferably pH 9.5.

During this hydrolytic phase, the temperature may be maintained at 60°C. for a longer period (say 3 hr) so as to reduce energy consumption ormay be maintained at 80-85° C. for a shorter period (say 1 hr).

The mixture is then conveyed (e.g. by an auger) to a trommel dryer 14which is provided with a counter-current airflow by an air fan or blower16 heated by heater 15. Preferably, it is at this point that optionaldehydrating materials (e.g. bentonite, zeolite, limestone, milled cerealgrain) are added to the dryer to facilitate the dehydration process.

The animal feed will normally exit the dryer 14 with about 10-11% (w/w)moisture content. This moisture content has been found to decrease toabout 7-8% (w/w) over 24-48 hr post-drying due to continued proteinhydrolysis.

The dried animal feed is conveyed to a hammer mill 17 for grinding to adesired granule/powder size and the granulated/powder feed material isconveyed to a packaging/transport area 18.

Where the product is to be used for direct feeding e.g. in fish feed,acid 19 may be added to the mixer 13 (after the initial mixing hasoccurred) or to the hammer mill 17 to reduce the pH of the final producte.g. to pH 6.5-7.5.

Where the product is to be mixed with acidic materials such as grain,the addition of acid may not be required as the alkaline product may bebalanced by the acidity of the grain.

It will be readily apparent to the skilled addressee that valuable feed,preferably for animals but also suitable for human consumption, can beproduced from animal tissue, substantially free of TDE contamination orhaving at least reduced likelihood of TDE contamination, and that theresultant feed is suitable for a wide range of potential uses, includinguse as a fertilizer.

So that the invention may be more readily understood and put intopractical effect, the skilled person is referred to the followingExamples.

EXAMPLE 1

Minced silverside was bought from a local butcher and 4×10 g aliquotsweighed out. Two of the aliquots were placed into metal beakers, spikedwith 1 mL 263K hamster scrapie crude brain homogeneate (cHs) and mixedthoroughly. To both beakers 20 mL of 5% calcium hydroxide solution wasadded, stirred, covered with foil and then placed in a 60° C. waterbathfor twelve hours. The solution was stirred approximately every 30minutes over the 12 hour incubation.

To the third 10 g aliquot of meat, 1 mL of cHs was added, mixedthoroughly and 20 mL of 1% SDS solution then added. This mixture wasplaced on an end-over-end mixer for 10 minutes. The mix was then boiledfor 5 minutes, clarified, aliquoted and frozen at −80° C. Thisconstitutes 287.1A cHs sample.

The final 10 g aliquot was placed into a metal beaker, spiked with 1 mLcHs, mixed thoroughly and then placed into the 60° C. oven to air-dry.Again, sample was mixed approximately every 30 minutes. Once dry, thedehydrated meat was removed from the oven and ground using a mortar andpestle. The ground meat was mixed with 20 ml of 1× Boiling Mix, placedon the end-over-end mixer, mixed for 12 hours at ambient temperature,then clarified and aliquoted. This constitutes the 287.1-1 cHs sample.

After 12 hours in the 60° C. waterbath, the two metal beakers wereremoved. One beaker was placed into the 60° C. oven and air-dried withintermittent stirring, as above. The material from the second beaker wasplaced into a centrifuge tube and the pH adjusted to pH of 7.5.22 mL of1× Boiling Mix was added, placed on the end-over-end and left to mix for12 hours at ambient temperature. This was then clarified, aliquoted andfrozen at −80° C. This constitutes the 287.1-2 cHs sample.

After dehydration at 60° C., the hydrolysed meat was removed from theoven and ground down using a mortar pestle. 20 mL of 50 mM sodiumacetate was added to the ground meat and the pH adjusted to pH 7.5. 23mL of 2× Boiling Mix was added and the tube placed on the end-over-endmixer to mix for 12 hours at ambient temperature. This was thenclarified, aliquoted and frozen at −80° C. This constitutes the 287.1-3cHs sample.

The pellet remaining from the 287.1-3cHs sample was resuspended andincubated with 10 mL of 50 mM sodium acetate buffer (pH 6.0), placed onthe end-over-end mixer for 60 minutes at ambient temperature. This wasthen clarified, aliquoted and frozen at −80° C. This constitutes the287.1-4 cHs sample.

Western blots were then performed on the samples. Western blot analysisof 263K hamster scrapie involves the use of the specific monoclonalantibody, 3F4. 3F4 recognizes both the normal cellular form of the priorprotein, PrP^(Sc), and the disease associated form, PrP^(Sc). Unlike thenormal cellular form, PrP^(Sc) is relatively protease resistant, and asproteinase K digestion of samples can be used to distinguish PrP^(Sc)from Proteinase K digestion of process samples is also useful forremoving proteins present in the process samples that could causenon-specific or cross-reactive background staining in the Western blotassay.

FIG. 2 shows a titration of the 263K hamster adapted scrapie stock usedin these studies (SP0172200). This shows the typical staining patternobserved for 263K using 3F4. The full length, mature PrP protein isbelieved to have an apparent molecular ratio M_(r)) of ˜33,000 daltons(33K, top band lane 2). Following proteinase K digestion, a predominantbroad band in the region of 28K, a fainter broad band ˜23K, and a sharpbut faint band ˜19K, are usually observed (see lane 1). Note that instocks of 263K Hs, these lower M_(r) species may also be detected (lane2), presumably due to endogenous proteases present in, or during thepreparation of the stock.

Lanes 4-10 show serial five fold dilutions of the 263K stock, from 1:25(10^(−1.4)) dilution to a 1:390625 (10^(−5.6)) dilution, respectively.The 28K PrP^(Sc) species can be detected in lanes 4-7, i.e. down to a10^(−3.5) dilution. On longer exposures of this blot (not shown), the28K species is also detected at 10^(−4.2) dilution. The end-point ortitre, of the stock is defined as the first dilution at which the 28KPrP species is no longer detected. The titre of this stock is therefore10^(−4.9), or 7.8×10⁴ arbitrary units/ml.

Western blot analysis of proteinase K digested process samples is shownin FIG. 3. Proteinase K digested 263K Hs stock SP0172200 serves as aninternal positive control (lane 1, 10^(−2.1) dilution). The 28K PrPspecies can be seen clearly in samples 287.1A and 287.1-1 (lanes 3 and4, respectively). A ˜33K protein species is also observed, suggestingthat proteinase K digestion of these samples is incomplete. No proteinbands are observed in samples 287.1-2, 287.1-3 or 287.1-4 (lanes 7-9,respectively), suggesting that the alkaline hydrolysis process hasremoved the PrP^(Sc) proteins. As no PrP^(Sc) species are observed forthese samples, they can be taken to have an end-point, or titre, of 10⁰,or 1×10⁰ arbitrary units/ml. Note that no estimate of the titre can bemade for the 287.1A or 287.1-1 samples.

To confirm that the PrP^(Sc) proteins had not simply been degraded bythe proteinase K digestion step, undigested process samples wereanalysed by Western blotting (FIG. 4). Both undigested and proteinase Kdigested 263K Hs stock SP0172200 served as internal positive controls(lanes 1 and 2, respectively). Again, PrP protein species can be seen insamples 287.1A and 287.1-1 (lanes 3 & 4, respectively). No protein bandsare evident in samples 287.1-2, 287.1-3 or 287.1-4 (lanes 7-9,respectively), confirming the results in FIG. 3.

EXAMPLE 2

Preliminary tests were performed to investigate the stability of pHfollowing treatment of meat material.

A 5% solution of calcium hydroxide was adjusted to pH 12.0 usinghydrochloric acid. 20 mL of this solution was then added to 10 g mincedlean beef and the sample mixed thoroughly. The pH was measured and thenmonitored over about 85 minutes during which time the pH remained at12.0.

Similarly, 5% calcium hydroxide was adjusted to pH 10.5 and 20 mL wasthoroughly mixed 10 g minced lean beef. The initial pH was 5.4 and thiswas adjusted to pH 10.5 by further addition of calcium hydroxide. The pHthen fell over the next 25 minutes to pH 10.3.

EXAMPLE 3

Two 10 g aliquots of minced silverside were placed into individual metalbeakers and each aliquot spiked with 1 mL of 263K hamster scrapie crudebrain homogenate (cHs). 20 mL of calcium hydroxide at pH 12.0 was thenadded to each beaker, the samples mixed thoroughly and the pH measuredas pH 12.0 for both samples. The beakers were then covered with foil andincubated at 75° C. for a total of 3 or 6 hrs (2 or 5 hrs in awaterbath, respectively, with the final 1 hr in an oven).

Four 10 g aliquots of minced silverside were placed into individualmetal beakers and each aliquot spiked with 1 mL of 263K hamster scrapiecrude brain homogenate (cHs). 18 mL of calcium hydroxide at pH 10.5 wasthen added to each beaker, the samples mixed thoroughly and the pHadjusted to pH 10.5 (with calcium hydroxide) for all samples. Thebeakers were then covered with foil and incubated at 60° C. for a totalof 3 hrs, 75° C. for a total of 3 or 6 hrs or 90° C. for 3 hrs(initially in a water bath with the final 1 hr in an oven).

All samples were neutralized to pH 6.5-7.5 on collection and thenadjusted to a final concentration of 1% SDS. The samples were mixedend-over-end for approximately 10 minutes at ambient temperature, boiledfor 5 minutes and clarified by low speed centrifugation. The supernatantfraction was then collected, aliquoted and stored at −70° C.

The details of the samples are, summarized in Table 1.

Samples were analyzed by Western blot.

As a control, one 10 g aliquot of minced silverside was spiked with 1 mLof 263K cHs, mixed thoroughly, and 20 ml of 1% SDS added. This samplewas mixed end-over-end for approximately 10 minutes at ambienttemperature, boiled for 5 minutes and clarified by low speedcentrifugation. The supernatant fraction was then collected, aliquotedand stored at −70° C.

FIG. 5 shows a titration of the 263K hamster adapted scrapie stock usedin these studies (SP0172200). This shows the typical staining patternobserved for 263K, using mAb 3F4. The full length, mature PrP protein isbelieved to have an apparent molecular ratio (M_(r)) of ˜33,000 daltons(33K, top band lane 2). Following proteinase K digestion, a predominantbroad band in the region of 28K, a fainter broad band ˜23K, and a sharpbut faint band ˜19K, are usually observed (see lane 1). Note that instocks of 263K Hs, these lower molecular weight species may also bedetected (lane 2), presumably due to endogenous proteases present in, orduring the preparation of the stock.

Processed samples were tested at neat dilution only, either withoutdigestion of following digestion with proteinase K at a finalconcentration of 10 μg/mL (based on previous results). The results areshown in FIGS. 6 and 7.

Proteinase K digested 263K cHs stock SP0172200 served as an internalpositive control (lane 1 of FIG. 6). The 28K PrP species can be seenclearly in sample 309.1A (both with and without proteinase K digestion;see lane 3 of FIGS. 6 and 7). A number of additional protein species areobserved in the undigested sample. The 33K PrP species is still visiblefollowing proteinase K digestion (lane 3 FIG. 6) suggesting thatproteinase K digestion was incomplete.

No protein bands were observed in any of the calcium hydroxide andheat-treated samples (lanes 5-10), either with or without proteinase Kdigestion, even on longer exposures of the Western blots (up to 30minutes). Thus, all of the calcium hydroxide and heat-treatment regimestested in this study removed the PrP^(Sc) proteins. As no PrP^(Sc)species are observed for these samples, they can be taken to have anend-point titre of 10⁰ or 10⁰ arbitrary units per mL. Notes that noestimate of the titre can be made for the 309.1A sample.

The level of PrP proteins present in the processed samples was notquantified by titration in the Western blot assay. However, although noPrP could be detected in any of the calcium hydroxide and heat-treatedsamples, an estimate of clearance in comparison to the estimated 263Kspike can be made.

The log₁₀ clearance factor is the ratio of the total amount of 263Kspiked into the starting meat material, to the total amount of 263Krecovered in the final, treated sample expressed as a log₁₀ value:

${Clearance} = \frac{{stock}\mspace{14mu}{titre} \times {spike}\mspace{14mu}{volume}}{{sample}\mspace{14mu}{titre} \times {sample}\mspace{14mu}{volume}}$

For all of the treated samples, a clearance factor of ≧3.4 logs wasachieved. These calculations are summarized in Table 2.

In conclusion, 263K Hs was spiked into samples of minced beef and theeffect of calcium hydroxide and heat-treatment according to the presentinvention measured in terms of PrP^(Sc) protein elimination. Thepositive results reported herein suggest that the method of theinvention is robust procedure for the removal or inactivation of TIDEfrom meat samples used in the production of animal feed.

It will be appreciated by the skilled person that the present inventionis not limited to the embodiments described in detail herein, and that avariety of other embodiments may be contemplated which are neverthelessconsistent with the broad spirit and scope of the invention.

All scientific and patent literature referred to in this specificationis incorporated herein by reference.

TABLE 1 Sample Description pH 309.1A Lean Minced beef spiked with 263KcHs (control) 6.5 309.1-1.3 Sample treated at pH 10.5, 75° C. for 3hours 8.8 309.1-1.6 Sample treated at pH 10.5, 75° C. for 6 hours 8.8309.1-2.3 Sample treated at pH 10.5, 60° C. for 3 hours n.d. 309.1-3.3Sample treated at pH 10.5, 90° C., for 3 hours 8.2 309.1-4.3 Sampletreated at pH 12.0, 75° C., for 3 hours 11.6 309.1-4.6 Sample treated atpH 12.0, 75° C., for 6 hours 11.5

TABLE 2 Titre Volume Total Clearance Sample (a.u per ml) (ml) (a.u perml) (log₁₀) Spike 7.8 × 10⁴   1 7.8 × 10⁴ — 309.1-1.3 cHs 1 × 10⁰ 30 3.0× 10¹ ≧3.41 309.1-1.6 cHs 1 × 10⁰ 25 2.5 × 10¹ ≧3.49 309.1-2.3 cHs 1 ×10⁰ 30 3.0 × 10¹ ≧3.41 309.1-3.3 cHs 1 × 10⁰ 26 2.6 × 10¹ ≧3.48309.1-4.3 cHs 1 × 10⁰ 20 2.0 × 10¹ ≧3.59 309.1-4.6 cHs 1 × 10⁰ 15 1.5 ×10¹ ≧3.72

1. A method of treating animal material to reduce or eliminate thepotential for TDE contamination, said method including the steps of: (i)adding alkali to animal material to maintain a pH of 8.5 to 13; and (ii)heating the material at step (i) to a temperature in the range 60° C. to99° to achieve alkali hydrolysis of protein present in the animalmaterial; (iii) dehydrating the material produced at step (ii) whereinthe duration of steps (i) and (ii) is 1 to 4 hours; wherein steps (i)and (ii) effectively sterilize the animal material with respect to anytransmissible degenerative encephalopathies present and wherein allsteps are performed at atmospheric pressure.
 2. The method of claim 1wherein the pH is at least 9.5 to
 13. 3. The method of claim 1 whereinthe temperature is 60° C.
 4. The method of claim 1 wherein a duration ofsteps (i) and (ii) is 1 to 3 hours.
 5. A method of producing afertilizer including the steps of: adding alkali to animal material tomaintain a pH of 8.5 to 13; and (ii) heating the material at step (i) toa temperature in the range of 60° C. to 99° to achieve alkali hydrolysisof protein present in the animal material; (iii) dehydrating thematerial produced at step (ii) wherein the duration of steps (i) and(ii) is 1 to 4 hours; wherein steps (i) and (ii) effectively sterilizethe animal material with respect to any transmissible degenerativeencephalopathies present and wherein all steps are performed atatmospheric pressure.
 6. The method of claim 5 wherein the pH is 9.5 to13.
 7. The method of claim 5 wherein the temperature is 60° C.
 8. Themethod of claim 5 wherein a duration of steps (i) and (ii) is 1 to 3hours.
 9. The method of claim 5 wherein a duration of steps (i) and (ii)is 1 to 2 hours.
 10. The method of claim 5 wherein the pH is in therange of 10.5 to
 13. 11. The method of claim 5 wherein the pH is in therange of 11.0 to 11.5.
 12. The method of claim 5 wherein the temperatureis in the range of 80° C. to 85° C.
 13. The method of claim 1 whereinthe pH is in the range of 10.5 to
 13. 14. The method of claim 1 whereinthe pH is in the range of 11.0 to 11.5.
 15. The method of claim 1wherein the temperature is in the range of 80° C. to 85° C.
 16. Themethod of claim 1 wherein a duration of steps (i) and (ii) is 1 to 2hours.